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High Temperature Storage of Spray-Dried Egg White1
High Temperature Storage of Spray-Dried Egg White1 2. ELECTROPHORETIC MOBILITY, CONALBUMIN-IRON COMPLEXING, SULFHYDRYL ACTIVITY, AND EVOLUTION OF VOLATILE BASES O. J. COTTEEILL, R. E. BALDWIN AND M. MYERS 2 Poultry Department and School of Home Economics, University of Missouri, Columbia, Missouri 65201 (Received for publication March 6, ,1967)
INTRODUCTION ITTLE attention has been given to the ' effect of this heat treatment on chemical and physical properties of the proteins in desugared egg white. This paper reports the effect of added yolk, pH of the liquid prior to spray-drying, and storage temperature of the electrophoretic mobility of the proteins, conalbumin-iron complexing, sulfhydryl activity and the evolution of volatile bases from spray-dried white. The influence of these treatments on functional properties was previously reported by Baldwin et al. (1967).
L
MATERIALS AND METHODS The procedures for the preparation of the egg white were the same as those described or cited by Hill et al. (196S) and by Baldwin et al. (1967). The sample pH levels referred to throughout the text represent the pH of the liquid prior to drying. The moisture content of the powder, storage conditions, and the pH of the reconstituted egg white solids were reported by Baldwin et al. (1967). The samples were reconstituted on a magnetic stirrer. Electrophoresis: The electrophoretic mobility of the spray-dried egg white after storage at elevated temperatures was compared with the unstored product. A rapid electrophoresis system with microporous cellulose polyacetate support strips was 1
Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 5091. Approved by Director. 2 Present address: Chico State College, Chico, California.
used according to the manufacturer's instructions (Gelman Instrument Co., 600 S. Wagner Rd., P. 0. Box 1448, Ann Arbor, Mich.). A barbital buffer (10.8 g. of sodium barbital and l.S g. of barbital made to 1000 ml. with distilled water, pH 8.6) was used. Each sample was reconstituted to 13% solids with distilled water before electrophoretic analysis. Four lambda of egg white were applied to the strips. Conalbumin Activity: Conalbumin actitivity was estimated by an unpublished method devised in this laboratory in 1964 by Seideman et al. which was a modification of the chemical assay technique of Fraenkel-Conrat and Feeney (1950). This procedure depends on the development of color by the conalbumin-iron complex. Conalbumin activity of the reconstituted powder was determined before and after 60 days of storage at each of the four elevated temperatures. The powder was first reconstituted with distilled water to a level of 12% solids. After the pH was adjusted to 5.0 with I N HC1, the egg white was clarified by filtration (Whatman . Filter Paper # 4 ) . Ten ml. of the filtrate was collected and adjusted to pH 8.0 with I N NaOH. Subsequently, after correcting for the amount of acid and base used to adjust the pH, the egg white preparation was diluted with 1% NaCl to one half of the original solids level. One ml. of a solution containing 0.537-mg. FeS0 4 -7H 2 0/ ml. was added to 20 ml. of this sample. After one hour, the absorbance at 450 mjjt,. was determined. Measurement of Volatile Bases: Volatile
1431
1432
0. J. COTTERILL, R. E. BALDWIN AND M.
bases evolved during storage were determined by placing 25 g. of each sample in a 250 ml. Erlenmeyer flask in an oven at 820,C. The volatile bases were carried from the flasks and were trapped in a standard sulfuric acid solution. By means of back titration, the amount of volatile bases was measured and expressed as the total milligrams of ammonia evolved during storage. The air was purified by serially passing it through powdered boric acid, flaked sodium hydroxide, and anhydrous calcium chloride traps. Cotton filters were installed throughout the system to prevent carryover of powder. Suljhydryl Activity: Sulfhydryl groups of reconstituted dried egg white (1.3% solids) were measured by the amperometric titration procedure of Benesch et al. (1955). The rotating platinum electrode was made according to the directions of Kolthoff and Harris (1946). A microburette was used to deliver the 0.001M AgN0 3 . To assure thorough mixing after each addition of AgN0 3 , a 1 minute equilibration time was allowed before reading the galvanometer. Reduced glutathione (Schwarz Laboratories, Inc., Mt. Vernon, N.Y.) served as a standard and was made every few days to equal 5 X 10~4M. One ml. of the reconstituted egg white (1.3% solids) was added to a buffer solution prepared from 4.0 ml. of 1.0M tris (hydroxymethylamino-methane), 3.4 ml. of l.ON HN0 3 , and 0.3 ml. of 1.0M KCl (pH 7.4 at 25°C), and the volume was brought to 30 ml. with deionized water. The specific end point of sulfhydryl activity was determined by extrapolation to zero (Kolthoff and Harris, 1946), and results were reported as meq. of AgN0 3 per 0.013 g. of egg white powder.
MYERS
conalbumin with iron, and of the egg white powders are reported herein. Also, the pattern of evolution of volatile bases during high temperature storage of the powder is shown. Systemic data on the solubility of the powder were not obtained. However, the powders prepared from the liquid egg white adjusted to high pH levels before storage at elevated temperatures were difficult to reconstitute. Some differences in the electropherograms and conalbumin-iron reaction data may reflect solubility changes of protein fractions. The coefficients of variation from the mean of the conalbumin and sulfhydryl activities of the unstored samples were 11.0% and 9.8%, respectively. These seeming wide variations can not be explained by the differences in the initial moisture content of the powders (Baldwin etal., 1967). Electrophoretic Mobility: The electropherograms obtained from the unstored and stored (60 days) egg white samples are shown in Figure 1. The ovomucoid fraction from the unstored samples, pH 5.0, did not resolve as well as with the higher pH powders. A similar tendency was indicated in the patterns published by Hill et al. (1965). It should be noted that the pH of the reconstituted egg white from this sample was about pH 5-6 while the others were in the region of pH 9-10 (Hill et al., 1965; Baldwin etal., 1967). Storage at 54°C. had no gross effects on the electropherograms of the reconstituted powders from liquid egg white adjusted to pH 5.0 and 6.5 prior to spray-drying. However, storage at 54°C. decreased the amount of mobile lysozyme of the powders reconstituted from the liquid egg at pH 8.5 and 9.5. The pH after reconstitution of the RESULTS AND DISCUSSION sample prepared from the pH 6.5 liquid The changes in the electrophoretic mo- tended to be slightly lower (pH 7.1-9.3) bility of the proteins complexing ability of than the liquid white adjusted to pH 8.5
STORAGE OF EGG WHITE POWDER
1433 Ovalbumins Ovomucoid Globulins Conalbumin Ovomucin
Unstored
Lysozyme
4
!:< »*.«ijt l
MMi:\
, .,,.. .
Stored 60 Days at 54° C.
Stored 60 Days at 60° C.
Stored 60 Days at 71° C.
Stored 60 Days at 82° C.
5.0
6.5
8.5
9.5
pH of Liquid Before Spray-Drying Fie. 1. Electrophoretic mobility of proteins from egg white solids which had been spray-dried from liquid adjusted to various pH levels and stored at high temperatures.
1434
O. J. COTTEEILL, R. E. BALDWIN AND M. MYERS
and 9.5 which reconstituted at pH 9.0-9.9. This change in lysozyme parallels that caused by heat treating liquid egg white at pH levels above 9.0 as reported by Seideman et al. (1963). Storing egg white solids at higher temperatures (60°, 71°, and 82°C.) caused progressively greater damage to the lysozyme, conalbumin, globulin, and ovomucoid components. The samples prepared from the pH 8.5 and 9.5 liquid egg white were affected more adversely than were the solids obtained from the pH 5.0-6.5 liquid. A residuum of the albumin fractions was the only component appearing on the electropherograms after storage for 60 days at 82°C. Conalbumin Activity: The amount of color (absorbance) produced by the conalbumin-iron complex after storage of the spray-dried egg white samples is shown in Table 1. Prior to high temperature storage of the powder, the ability of the conalbumin fraction to react with iron was affected little by the pH of the liquid before drying and/or the presence of yolk. Storage at 54°C. tended to enhance the intensity"of the conalbumin-iron reaction. The de-
creased activity for the yolk-free, pH 9.5 sample stored at 54°C. is probably erroneous. Storage at the higher temperatures had a negative effect on this reaction. The presence of yolk in the powder did not cause any consistent trends resulting from the heat treatments. In general, the stored powder prepared from the liquid of the high pH values had less conalbumin activity than the solids from the liquid at low pH. It appears that in the dry state, the heat damage is greater at high pH. (9-10), while heat damage to conalbumin in liquid egg white is greater at a low pH level (6-8) near its isoelectric point (Seideman et al., 1963; Hill et al., 1965; and Cunningham and Lineweaver, 1965). Figure 2 illustrates differences in appearance of solutions resulting from the ability of the conalbumin fraction of spray-dried egg white to react with iron. The clearer solutions at high pH levels and high temperatures indicate decreased conalbumin activity. Solutions from the conalbumin test on spray-dried white stored at 54° and 60°C. were not included because visual differences were less apparent. Sulfhydryl Activity: No trends were ob-
TABLE 1.—Absorbance1 ofiron-conalbumin complex from spray-dried egg white after storage at high temperatures Temperature and Time of storage
Sample description Yolk2
5.0 5.0
1 2
'
54°C.
pH before drying
60°C. .
71°C.
82°C.
Unstored
Stored 60 days
Unstored
Stored 60 days
Unstored
Stored 60 days
Unstored
Stored 60 days
free added
.298 .292
.332 .320
.336 .334
.292 .306
.306 .272
.233 .184
.392 .352
.025 .029
6.5 6.5
free added
.272 .292
.332 .322
.300 .332
.148 .276
.248 .270
.178 .186
.362 .368
.033 .020
8.5 8.5
free added
.314 .288
.300 .264
.342 .342
.148 .238
.284 .302
.084 .122
.392 .346
.019 .019
9.5 9.5
free added
.302 .296
.324 .268
.324 .320
.180 .232
.298 .302
.019 .084
.332 .323
.016 .022
Absorbance at 450 mji. Average of duplicate observations. Yolk added at 0.03% level on a liquid basis.
1435
STORAGE OF EGG WHITE POWDER
Unstored
Stored 60 Days at 71° C
_J
Lai -v
W V
' Stored 60 Days at 82° C
h
g3—rijj
Smmtss»
V Yolk Yolk free added 5.0
Yolk Yolk free added 6.5
Yolk Yolk free added 8.5
Yolk free
Yolk added 9.5
pH of Liquid Before Spray-Drying FIG. 2. Appearance of iron-conalbumin complex from spray-dried egg white after storage at high temperatures.
served in the sulfhydryl activity of the spray-dried egg white samples stored at high temperatures (Table 2). With a few exceptions, the values ranged between O.S5-0.71 meq. AgN0 3 per 0.013 g. of
powder. These data indicate that the degradation mechanism of egg white solids at high temperatures does not involve sulfhydryl groups. Many of the samples held at 82°C. reconstituted as a gelatinous mass.
1436
O. J. COTTEEILL, R. E. BALDWIN AND M.
MYERS
TABLE 2.—Sulfhydryl activity1 of reconstituted spray-dried egg white
after storage at high temperatures
Sample description
Temperature and time of storage 54°C.
60°C.
71°C.
82°C/
Un- Stored stored 60 days
UnStored stored 60 days
Un- Stored stored 60 days
UnStored stored 60 days
pH before drying
Yolk2
5.0 5.0
free added
.55 .67
.39 .71
.68 .67
.64 .71
.66 .48
.68 .66
.67 .65
.61 .62
6.5 6.5
free added
.66 .67
.38 .65
.72 .70
.66 .65
.61 .64
.61 .60
.60 .67
—
8.5 8.5
free added
.68 .62
.62 .62
.63 .61
.61
.46 .54
.67 .58
.58 .63
—
9.5 9.5
free added
.64 .62
.54 .61
.57 .57
.56 .55
.65 .58
.37 .48
.56 .60
1 2
—
Milliequivalents of AgNC>3 per 0.013 g. of egg white powder. Average of duplicate observations. Yolk added at 0.03% on a liquid basis.
Hence, sulfhydryl measurements were not possible. Volatile Bases: A strong ammoniacal odor was detected when portions of the dried egg white were removed periodically from the sealed glass containers. This odor was particularly noticeable with egg white solids held at 82 °C. It is assumed that the main constitutent of this volatile material was ammonia. The pattern of evolution of
Storage Time (days)
FIG. 3. Evolution of volatile bases from egg white solids which had been spray-dried from liquid adjusted to four pH levels and stored at 82°C.
volatile compounds from the egg white powders prepared by spray-drying liquid at different pH levels is shown in Figure 3. Ammonia release was slightly more rapid during the first few days of the 14-day storage period than during the last. Also, the rate of ammonia evolution was greater from the powders spray-dried from liquid adjusted to pH 6.5, 8.5 and 9.5 than from the powder obtained from liquid at pH 5.0. The powder prepared from the liquid at pH 6.5 evolved ammonia almost as fast as did the powders from the liquid at pH 8.5 and 9.5. This seeming fast rate of evolution can be explained by the high pH of the reconstituted powder (near pH 9.0) prepared from the liquid at pH 6.5 (Hill et al., 1965; Baldwin et al., 1967). Also high temperature storage tended to reduce the pH of the reconstituted powders which were spray-dried from liquid of pH 6.5, 8.5, and 9.5. The data suggest that the mechanism of degradation of egg white solids stored at high temperatures involves nitrogenous constituents of the proteins. The presence of added yolk did not affect the rate of evolution of volatile bases.
STORAGE OF EGG WHITE POWDER SUMMARY
Spray-dried egg white, adjusted to pH levels of 5.0, 6.5, 8.5, and 9.5 before drying, was stored for 60 days at 54°, 60°, 71°, or 82°C. Both yolk free and spraydried egg white with yolk added (0.03% on a liquid basis) were studied at each pH level. Changes which occurred during the storage periods were evaluated by electrophoretic mobility, iron-conalbumin complexing ability, sulfhydryl activity and evolution of volatile bases. Degradative changes such as decreased lysozyme mobility, iron-conalbumin complexing, and solubility as well as increased evolution of ammonia during storage at high temperatures were enhanced by high pH of the egg solids. Electrophoretic analysis indicated that ovalbumin was more stable than the other protein fractions. No trends were evident in sulfhydryl activity of spray-dried egg white subjected to the elevated storage temperatures. The addition of yolk to egg white prior to drying did not affect any of the chemical or physical observations. ACKNOWLEDGMENT
The authors appreciate the technical assistance of Jill Matter, Walter Seideman, Ruth Upchurch, and Marion Vogt whose
1437
cooperation and help made this report possible. REFERENCES Baldwin, R. E., O. J. Cotterill, M. Martinek Thompson and M. Meyers, 1967. High temperature storage of spray-dfied egg white. I. Whipping time and quality of angel cake. Poultry Sci. 46: 1421-1430. Banwart, G. J., and J. C. Ayres, 1956. The effect of high temperature storage on the content of Salmonella on the functional properties of dried egg white. Food Technol. 10: 68-73. Benesch, R. E., H. A. Lardy and R. Benesch, 19SS. The sulfhydryl groups of crystalline proteins. I. Some albumins, enzymes, and hemoglobins. J. Biol. Chem. 216: 663-676. Cunningham, F. E., and H. Lineweaver, 1965. Stabilization of egg-white proteins to pasteurizing temperatures above 60° C. Food Technol. 19: 136-141. Fraenkel-Conrat, H., and R. E. Feeney, 1950. The metal-binding activity of conalbumin. Arch. Biochem. 29: 101-113. Hill, W. M., O. J. Cotterill, E. M. Funk and R. E. Baldwin, 1965. Spray-drying egg white at various pH levels. Poultry Sci. 44: 1155-1163. Kolthoff, I. M., and W. E. Harris, 1946. Amperometric titration of mercaptans with silver nitrate. Ind. Eng. Chem. 18: 161-162. Seideman, W. E., O. J. Cotterill and E. M. Funk, 1963. Factors affecting heat coagulation of egg white. Poultry Sci. 42: 406-417. Seideman, W. E., W. M. Hill and O. J. Cotterill, 1964. Colorimetric determination of conalbumin activity in egg white. Unpublished, Poultry Dept, University of Missouri, Columbia.
NEWS AND NOTES (Continued front page 1430) ever, if the immediate needs of I.P.B. are to be met, it will almost certainly be necessary to enlist the aid of emeritus biologists for many aspects of the work. A recommendation to this effect was made by Dr. John R. Olive, Executive Director of A.I.B.S., to the House Subcommittee on Science, Research and Technology. The utilization of emeritus biologists in significant positions in teaching, research and administration has been fostered through the American Institute of Biological Sciences Emeritus Biologists Program which is now in its third year.
Many opportunities are still available to emeriti to replace biologists on sabbatical and for full or part-time teaching positions on a year-to-year basis. Through this Program, emeritus biologists have found positions in almost all regions of the United States and in overseas assignments. If you are retiring or planning to retire within a year or two, and are seriously considering continuing your active professional life after retirement let the A.I.B.S. Emeritus Biologists program assist you in obtaining a challenging professional appointment.
(Continued on page 1442)
INTRODUCTION ITTLE attention has been given to the ' effect of this heat treatment on chemical and physical properties of the proteins in desugared egg white. This paper reports the effect of added yolk, pH of the liquid prior to spray-drying, and storage temperature of the electrophoretic mobility of the proteins, conalbumin-iron complexing, sulfhydryl activity and the evolution of volatile bases from spray-dried white. The influence of these treatments on functional properties was previously reported by Baldwin et al. (1967).
L
MATERIALS AND METHODS The procedures for the preparation of the egg white were the same as those described or cited by Hill et al. (196S) and by Baldwin et al. (1967). The sample pH levels referred to throughout the text represent the pH of the liquid prior to drying. The moisture content of the powder, storage conditions, and the pH of the reconstituted egg white solids were reported by Baldwin et al. (1967). The samples were reconstituted on a magnetic stirrer. Electrophoresis: The electrophoretic mobility of the spray-dried egg white after storage at elevated temperatures was compared with the unstored product. A rapid electrophoresis system with microporous cellulose polyacetate support strips was 1
Contribution from the Missouri Agricultural Experiment Station. Journal Series Number 5091. Approved by Director. 2 Present address: Chico State College, Chico, California.
used according to the manufacturer's instructions (Gelman Instrument Co., 600 S. Wagner Rd., P. 0. Box 1448, Ann Arbor, Mich.). A barbital buffer (10.8 g. of sodium barbital and l.S g. of barbital made to 1000 ml. with distilled water, pH 8.6) was used. Each sample was reconstituted to 13% solids with distilled water before electrophoretic analysis. Four lambda of egg white were applied to the strips. Conalbumin Activity: Conalbumin actitivity was estimated by an unpublished method devised in this laboratory in 1964 by Seideman et al. which was a modification of the chemical assay technique of Fraenkel-Conrat and Feeney (1950). This procedure depends on the development of color by the conalbumin-iron complex. Conalbumin activity of the reconstituted powder was determined before and after 60 days of storage at each of the four elevated temperatures. The powder was first reconstituted with distilled water to a level of 12% solids. After the pH was adjusted to 5.0 with I N HC1, the egg white was clarified by filtration (Whatman . Filter Paper # 4 ) . Ten ml. of the filtrate was collected and adjusted to pH 8.0 with I N NaOH. Subsequently, after correcting for the amount of acid and base used to adjust the pH, the egg white preparation was diluted with 1% NaCl to one half of the original solids level. One ml. of a solution containing 0.537-mg. FeS0 4 -7H 2 0/ ml. was added to 20 ml. of this sample. After one hour, the absorbance at 450 mjjt,. was determined. Measurement of Volatile Bases: Volatile
1431
1432
0. J. COTTERILL, R. E. BALDWIN AND M.
bases evolved during storage were determined by placing 25 g. of each sample in a 250 ml. Erlenmeyer flask in an oven at 820,C. The volatile bases were carried from the flasks and were trapped in a standard sulfuric acid solution. By means of back titration, the amount of volatile bases was measured and expressed as the total milligrams of ammonia evolved during storage. The air was purified by serially passing it through powdered boric acid, flaked sodium hydroxide, and anhydrous calcium chloride traps. Cotton filters were installed throughout the system to prevent carryover of powder. Suljhydryl Activity: Sulfhydryl groups of reconstituted dried egg white (1.3% solids) were measured by the amperometric titration procedure of Benesch et al. (1955). The rotating platinum electrode was made according to the directions of Kolthoff and Harris (1946). A microburette was used to deliver the 0.001M AgN0 3 . To assure thorough mixing after each addition of AgN0 3 , a 1 minute equilibration time was allowed before reading the galvanometer. Reduced glutathione (Schwarz Laboratories, Inc., Mt. Vernon, N.Y.) served as a standard and was made every few days to equal 5 X 10~4M. One ml. of the reconstituted egg white (1.3% solids) was added to a buffer solution prepared from 4.0 ml. of 1.0M tris (hydroxymethylamino-methane), 3.4 ml. of l.ON HN0 3 , and 0.3 ml. of 1.0M KCl (pH 7.4 at 25°C), and the volume was brought to 30 ml. with deionized water. The specific end point of sulfhydryl activity was determined by extrapolation to zero (Kolthoff and Harris, 1946), and results were reported as meq. of AgN0 3 per 0.013 g. of egg white powder.
MYERS
conalbumin with iron, and of the egg white powders are reported herein. Also, the pattern of evolution of volatile bases during high temperature storage of the powder is shown. Systemic data on the solubility of the powder were not obtained. However, the powders prepared from the liquid egg white adjusted to high pH levels before storage at elevated temperatures were difficult to reconstitute. Some differences in the electropherograms and conalbumin-iron reaction data may reflect solubility changes of protein fractions. The coefficients of variation from the mean of the conalbumin and sulfhydryl activities of the unstored samples were 11.0% and 9.8%, respectively. These seeming wide variations can not be explained by the differences in the initial moisture content of the powders (Baldwin etal., 1967). Electrophoretic Mobility: The electropherograms obtained from the unstored and stored (60 days) egg white samples are shown in Figure 1. The ovomucoid fraction from the unstored samples, pH 5.0, did not resolve as well as with the higher pH powders. A similar tendency was indicated in the patterns published by Hill et al. (1965). It should be noted that the pH of the reconstituted egg white from this sample was about pH 5-6 while the others were in the region of pH 9-10 (Hill et al., 1965; Baldwin etal., 1967). Storage at 54°C. had no gross effects on the electropherograms of the reconstituted powders from liquid egg white adjusted to pH 5.0 and 6.5 prior to spray-drying. However, storage at 54°C. decreased the amount of mobile lysozyme of the powders reconstituted from the liquid egg at pH 8.5 and 9.5. The pH after reconstitution of the RESULTS AND DISCUSSION sample prepared from the pH 6.5 liquid The changes in the electrophoretic mo- tended to be slightly lower (pH 7.1-9.3) bility of the proteins complexing ability of than the liquid white adjusted to pH 8.5
STORAGE OF EGG WHITE POWDER
1433 Ovalbumins Ovomucoid Globulins Conalbumin Ovomucin
Unstored
Lysozyme
4
!:< »*.«ijt l
MMi:\
, .,,.. .
Stored 60 Days at 54° C.
Stored 60 Days at 60° C.
Stored 60 Days at 71° C.
Stored 60 Days at 82° C.
5.0
6.5
8.5
9.5
pH of Liquid Before Spray-Drying Fie. 1. Electrophoretic mobility of proteins from egg white solids which had been spray-dried from liquid adjusted to various pH levels and stored at high temperatures.
1434
O. J. COTTEEILL, R. E. BALDWIN AND M. MYERS
and 9.5 which reconstituted at pH 9.0-9.9. This change in lysozyme parallels that caused by heat treating liquid egg white at pH levels above 9.0 as reported by Seideman et al. (1963). Storing egg white solids at higher temperatures (60°, 71°, and 82°C.) caused progressively greater damage to the lysozyme, conalbumin, globulin, and ovomucoid components. The samples prepared from the pH 8.5 and 9.5 liquid egg white were affected more adversely than were the solids obtained from the pH 5.0-6.5 liquid. A residuum of the albumin fractions was the only component appearing on the electropherograms after storage for 60 days at 82°C. Conalbumin Activity: The amount of color (absorbance) produced by the conalbumin-iron complex after storage of the spray-dried egg white samples is shown in Table 1. Prior to high temperature storage of the powder, the ability of the conalbumin fraction to react with iron was affected little by the pH of the liquid before drying and/or the presence of yolk. Storage at 54°C. tended to enhance the intensity"of the conalbumin-iron reaction. The de-
creased activity for the yolk-free, pH 9.5 sample stored at 54°C. is probably erroneous. Storage at the higher temperatures had a negative effect on this reaction. The presence of yolk in the powder did not cause any consistent trends resulting from the heat treatments. In general, the stored powder prepared from the liquid of the high pH values had less conalbumin activity than the solids from the liquid at low pH. It appears that in the dry state, the heat damage is greater at high pH. (9-10), while heat damage to conalbumin in liquid egg white is greater at a low pH level (6-8) near its isoelectric point (Seideman et al., 1963; Hill et al., 1965; and Cunningham and Lineweaver, 1965). Figure 2 illustrates differences in appearance of solutions resulting from the ability of the conalbumin fraction of spray-dried egg white to react with iron. The clearer solutions at high pH levels and high temperatures indicate decreased conalbumin activity. Solutions from the conalbumin test on spray-dried white stored at 54° and 60°C. were not included because visual differences were less apparent. Sulfhydryl Activity: No trends were ob-
TABLE 1.—Absorbance1 ofiron-conalbumin complex from spray-dried egg white after storage at high temperatures Temperature and Time of storage
Sample description Yolk2
5.0 5.0
1 2
'
54°C.
pH before drying
60°C. .
71°C.
82°C.
Unstored
Stored 60 days
Unstored
Stored 60 days
Unstored
Stored 60 days
Unstored
Stored 60 days
free added
.298 .292
.332 .320
.336 .334
.292 .306
.306 .272
.233 .184
.392 .352
.025 .029
6.5 6.5
free added
.272 .292
.332 .322
.300 .332
.148 .276
.248 .270
.178 .186
.362 .368
.033 .020
8.5 8.5
free added
.314 .288
.300 .264
.342 .342
.148 .238
.284 .302
.084 .122
.392 .346
.019 .019
9.5 9.5
free added
.302 .296
.324 .268
.324 .320
.180 .232
.298 .302
.019 .084
.332 .323
.016 .022
Absorbance at 450 mji. Average of duplicate observations. Yolk added at 0.03% level on a liquid basis.
1435
STORAGE OF EGG WHITE POWDER
Unstored
Stored 60 Days at 71° C
_J
Lai -v
W V
' Stored 60 Days at 82° C
h
g3—rijj
Smmtss»
V Yolk Yolk free added 5.0
Yolk Yolk free added 6.5
Yolk Yolk free added 8.5
Yolk free
Yolk added 9.5
pH of Liquid Before Spray-Drying FIG. 2. Appearance of iron-conalbumin complex from spray-dried egg white after storage at high temperatures.
served in the sulfhydryl activity of the spray-dried egg white samples stored at high temperatures (Table 2). With a few exceptions, the values ranged between O.S5-0.71 meq. AgN0 3 per 0.013 g. of
powder. These data indicate that the degradation mechanism of egg white solids at high temperatures does not involve sulfhydryl groups. Many of the samples held at 82°C. reconstituted as a gelatinous mass.
1436
O. J. COTTEEILL, R. E. BALDWIN AND M.
MYERS
TABLE 2.—Sulfhydryl activity1 of reconstituted spray-dried egg white
after storage at high temperatures
Sample description
Temperature and time of storage 54°C.
60°C.
71°C.
82°C/
Un- Stored stored 60 days
UnStored stored 60 days
Un- Stored stored 60 days
UnStored stored 60 days
pH before drying
Yolk2
5.0 5.0
free added
.55 .67
.39 .71
.68 .67
.64 .71
.66 .48
.68 .66
.67 .65
.61 .62
6.5 6.5
free added
.66 .67
.38 .65
.72 .70
.66 .65
.61 .64
.61 .60
.60 .67
—
8.5 8.5
free added
.68 .62
.62 .62
.63 .61
.61
.46 .54
.67 .58
.58 .63
—
9.5 9.5
free added
.64 .62
.54 .61
.57 .57
.56 .55
.65 .58
.37 .48
.56 .60
1 2
—
Milliequivalents of AgNC>3 per 0.013 g. of egg white powder. Average of duplicate observations. Yolk added at 0.03% on a liquid basis.
Hence, sulfhydryl measurements were not possible. Volatile Bases: A strong ammoniacal odor was detected when portions of the dried egg white were removed periodically from the sealed glass containers. This odor was particularly noticeable with egg white solids held at 82 °C. It is assumed that the main constitutent of this volatile material was ammonia. The pattern of evolution of
Storage Time (days)
FIG. 3. Evolution of volatile bases from egg white solids which had been spray-dried from liquid adjusted to four pH levels and stored at 82°C.
volatile compounds from the egg white powders prepared by spray-drying liquid at different pH levels is shown in Figure 3. Ammonia release was slightly more rapid during the first few days of the 14-day storage period than during the last. Also, the rate of ammonia evolution was greater from the powders spray-dried from liquid adjusted to pH 6.5, 8.5 and 9.5 than from the powder obtained from liquid at pH 5.0. The powder prepared from the liquid at pH 6.5 evolved ammonia almost as fast as did the powders from the liquid at pH 8.5 and 9.5. This seeming fast rate of evolution can be explained by the high pH of the reconstituted powder (near pH 9.0) prepared from the liquid at pH 6.5 (Hill et al., 1965; Baldwin et al., 1967). Also high temperature storage tended to reduce the pH of the reconstituted powders which were spray-dried from liquid of pH 6.5, 8.5, and 9.5. The data suggest that the mechanism of degradation of egg white solids stored at high temperatures involves nitrogenous constituents of the proteins. The presence of added yolk did not affect the rate of evolution of volatile bases.
STORAGE OF EGG WHITE POWDER SUMMARY
Spray-dried egg white, adjusted to pH levels of 5.0, 6.5, 8.5, and 9.5 before drying, was stored for 60 days at 54°, 60°, 71°, or 82°C. Both yolk free and spraydried egg white with yolk added (0.03% on a liquid basis) were studied at each pH level. Changes which occurred during the storage periods were evaluated by electrophoretic mobility, iron-conalbumin complexing ability, sulfhydryl activity and evolution of volatile bases. Degradative changes such as decreased lysozyme mobility, iron-conalbumin complexing, and solubility as well as increased evolution of ammonia during storage at high temperatures were enhanced by high pH of the egg solids. Electrophoretic analysis indicated that ovalbumin was more stable than the other protein fractions. No trends were evident in sulfhydryl activity of spray-dried egg white subjected to the elevated storage temperatures. The addition of yolk to egg white prior to drying did not affect any of the chemical or physical observations. ACKNOWLEDGMENT
The authors appreciate the technical assistance of Jill Matter, Walter Seideman, Ruth Upchurch, and Marion Vogt whose
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cooperation and help made this report possible. REFERENCES Baldwin, R. E., O. J. Cotterill, M. Martinek Thompson and M. Meyers, 1967. High temperature storage of spray-dfied egg white. I. Whipping time and quality of angel cake. Poultry Sci. 46: 1421-1430. Banwart, G. J., and J. C. Ayres, 1956. The effect of high temperature storage on the content of Salmonella on the functional properties of dried egg white. Food Technol. 10: 68-73. Benesch, R. E., H. A. Lardy and R. Benesch, 19SS. The sulfhydryl groups of crystalline proteins. I. Some albumins, enzymes, and hemoglobins. J. Biol. Chem. 216: 663-676. Cunningham, F. E., and H. Lineweaver, 1965. Stabilization of egg-white proteins to pasteurizing temperatures above 60° C. Food Technol. 19: 136-141. Fraenkel-Conrat, H., and R. E. Feeney, 1950. The metal-binding activity of conalbumin. Arch. Biochem. 29: 101-113. Hill, W. M., O. J. Cotterill, E. M. Funk and R. E. Baldwin, 1965. Spray-drying egg white at various pH levels. Poultry Sci. 44: 1155-1163. Kolthoff, I. M., and W. E. Harris, 1946. Amperometric titration of mercaptans with silver nitrate. Ind. Eng. Chem. 18: 161-162. Seideman, W. E., O. J. Cotterill and E. M. Funk, 1963. Factors affecting heat coagulation of egg white. Poultry Sci. 42: 406-417. Seideman, W. E., W. M. Hill and O. J. Cotterill, 1964. Colorimetric determination of conalbumin activity in egg white. Unpublished, Poultry Dept, University of Missouri, Columbia.
NEWS AND NOTES (Continued front page 1430) ever, if the immediate needs of I.P.B. are to be met, it will almost certainly be necessary to enlist the aid of emeritus biologists for many aspects of the work. A recommendation to this effect was made by Dr. John R. Olive, Executive Director of A.I.B.S., to the House Subcommittee on Science, Research and Technology. The utilization of emeritus biologists in significant positions in teaching, research and administration has been fostered through the American Institute of Biological Sciences Emeritus Biologists Program which is now in its third year.
Many opportunities are still available to emeriti to replace biologists on sabbatical and for full or part-time teaching positions on a year-to-year basis. Through this Program, emeritus biologists have found positions in almost all regions of the United States and in overseas assignments. If you are retiring or planning to retire within a year or two, and are seriously considering continuing your active professional life after retirement let the A.I.B.S. Emeritus Biologists program assist you in obtaining a challenging professional appointment.
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